BioResearch Bio Research Stem Cell Research Tools for Biologically Relevant Results BioResearch Stem Cell Products It All Stems from Here – Simplify Your Reprogramming, Proliferation “I can’t find a reliable solution for generation and expansion of iPSCs suited for research and clinical applications” SOLUTION: The new L7™ hiPSC Reprogramming and hPSC Culture System provides a complete, xenofree solution from reprogramming to growth and expansion. Page 5 iPSC Workflow “How can I feed my hPSCs every other day while still maintaining normal karyotype and differentiation potential?” SOLUTION: Lonza now offers the L7™ hPSC Culture System - a xenofree, defined system that allows for every other day feeding of hPSCs. Page 8 PSC Culture 2 and Differentiation “How can I stably modulate gene expression in pluripotent stem cells?” SOLUTION: Our non-viral Nucleofector™ Technology allows simple and reliable genome editing of ESCs, iPSCs or other cells using ZFN, TALEN or CRISPR/Cas9 technologies. Page 11 Genome Editing “How can I get the full differentiation potential from my stem cells?” SOLUTION: Lonza stem cells have high purity and homogeneity (confirmed by FACS analysis of positive and negative markers). Page 15 Adult Stem Cells 3 BioResearch Stem Cell Products Your Stem Cell Research Toolkit perior results. We supply high-quality primary and stem cell-derived cell types, with optimized medium and proven protocols; superior transfection technology (even for the most challenging cell types), and a wide range of cell analysis tools. Lonza offers convenient solutions to help scientists overcome a variety of stem cell research challenges whether working with adult stem cells or pluripotent stem cells. Our integrated products are optimized and tested together to provide su- Pluripotent Stem Cells Adult Stem Cells Cell Analysis Tools Reprogramming – Nucleofector™ Technology for efficient reprogramming of e.g. fibroblasts, PBMCs, CD34 – L7™ PBMC Reprogramming Bundle for reliable generation of iPSCs from mononuclear cells Proliferation – Broad collection of adult stem cells, progenitors or precursors with optimized growth media, e.g. mesenchymal, adipose-derived or dental pulp stem cells as well as pre-adipocytes, hematopoietic cells and neural progenitors Living Cell Imaging – CytoSMART™ System: small, easy and affordable life cell imaging system for documentation of iPSC generation or monitoring of stem cells cultured under hypoxic conditions Proliferation – L7™ hPSC Culture System – Lonza’s pluripotent services Differentiation and Modification – Nucleofector™ Technology for genome editing in ESCs or iPSCs using ZFN, TALEN and CRISPR systems – PSC derived primary cells Differentiation and Modification – Specialized media for adipose, chondrogenic and osteogenic differentiation of human MSCs – Nucleofector™ Technology with optimized protocols for transfection of primary adult stem cells, e.g. human MSC, human ADSCs, rat/mouse NSCs 4 Cell Analysis Tools – Collection of tools to analyze viability or functionality of stem cells or stem cell-derived cells Pluripotent Stem Cells – Reprogramming L7™ hiPSC Reprogramming and hPSC Culture System “I can’t find a reliable solution for generation and expansion of iPSCs suited for research and clinical applications” In 2014 Lonza introduced its new L7™ hiPSC Reprogramming and hPSC Culture System for basic stem-cell research, disease modeling, drug development and regenerative medicine. Our offering includes innovative tools to simplify and streamline the stem-cell culturing and reprogramming workflow. The novel L7™ PSC Culture System supports the generation of reproducible, clinically relevant data as a solid basis for more efficient drug development and accelerated progress in regenerative medicine. SOLUTION: The new L7™ hiPSC Reprogramming and hPSC Culture System provides a complete, xeno-free solution from reprogramming to growth and expansion. ZFN, TALEN or CRISPR. The L7™ hiPSC Reprogramming and hPSC Culture System includes seven key components that are aligned and function as a robust workflow. This novel culture system is the only available complete solution for the generation of induced pluripotent stem-cells (iPSCs), the maintenance of human embryonic stem cells (hESCs) and iPSCs under defined, xeno-free conditions. The culture system allows for every other day feeding and supports long term culture and maintenance of hPSCs. The L7™ hiPSC Reprogramming and hPSC Culture System thus provides a seamless transition from the lab bench to clinical development applications. www.lonza.com /L7 – for further information 1 Workflow 3 7 Components Nucleofector™ Technology 1 Provider Clonetics™ and Poietics™ Primary Cells 1 4 7 L7™ hPSC Medium BulletKit™ 5 L7™ Reprogramming Bundles Reviving & Priming 2 L7™ hPSC Passaging Solution Reprogramming L7™ hPSC Cryosolution L7™ hPSC Matrix 6 Colony Appearance 5 Colony Selection & Expansion Banking & Characterization BioResearch Stem Cell Products Pluripotent Stem Cells – Reprogramming Nucleofector™ Technology - For iPSC Generation Transfection Results for Typical Reprogramming Starter Cells The Nucleofector™ Technology has been demonstrated to be an efficient and cost-effective non-viral alternative for iPSC generation and is being used by leading scientists around the world. Efficiency* Viability* Kit for 4D-Nucleofector™ and 96-well Shuttle™ Systems Nucleofection benefits for reprogramming: Kit for Nucleofector™ II/2b Device Primary Cells – Simple, single-step procedure to introduce DNA/RNA – Successfully tested for generation of iPSCs from various cell types, including PBMCs and fibroblasts – NEW - Fully defined protocol for PBMC reprogramming using the 4D-Nucleofector™ System (L7™ PBMC Reprogramming Bundle) – Seamless workflow in combination with our L7™ hPSC Culture System for feeder- and xeno-free iPSC culture – Availability of 4D-Nucleofector™ Kits with cGMP Solutions Adipose-derived 73–94% stem cells, human (ADSC) 58–85% P1 Primary Cell Human MSC CD34+ cells, human 66–83% 62–70% P3 Primary Cell Human CD34 Fibroblasts 40–95% P2 Primary Cell Human Fibroblast Keratinocytes 40–80% P3 Primary Cell Human Keratinocyte PBMCs 30–95% P3 Primary Cell Human T Cell www.lonza.com /celldatabase – for further information and protocol guidance L7™ PBMC Reprogramming Bundle Using the 4D-Nucleofector™ System, Lonza’s Pluripotent Stem Cell Innovation Team has developed an optimized protocol for reprogramming of PBMCs (erythroblast population) using episomal vectors. Components required to run the optimized protocol: – Lonza Human Peripheral Blood Mononuclear Cells as positive control donor – NEW - L7™ hPSC Reprogramming Bundle – L7™ PBMC Priming-Recovery Basal Medium for priming of PBMCs towards erythroblasts prior to reprogramming and optimal recovery post transfection – L7™ PBMC Reprogramming Enhancers A and B for optimal reprogramming results – 4D-Nucleofector™ X Unit – P3 Primary Cell 4D-Nucleofector™ Kit 6 BioResearch Stem Cell Products Pluripotent Stem Cells – Reprogramming Case Study Lonza L7™ hiPSC Reprogramming and hPSC Culture System By Ha T. Tran1, Rex E. Lacambacal1,2, Candace L. Lynch1, Jeanne F. Loring1, Inbar Friedrich Ben-Nun3, Yu-Chieh Wang1,4 1) Department of Chemical Physiology, Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA; 2) California State University, Channel Islands, CA, USA; 3) Lonza Walkersville, Inc., Walkersville, MD, USA; 4) Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, TX, USA In this study, the authors evaluated the Lonza protocol for a certain individual – referred to as individual #418 – for which various reprogramming attempts had failed thus far. Previously, they successfully generated hiPSCs from primary cells of numerous individuals using retro- or Sendai virus-mediated methods. However, in the case of individual #418, these approaches failed to yield colonies following transduction attempts with both dermal fibroblasts from several biopsies and epithelial cells from urine samples. Using the Lonza L7™ hiPSC Reprogramming and hPSC Culture System, they have generated hiPSCs from PBMCs of individual #418. The data presented in the article demonstrate that the Lonza protocol can be used for the generation of high quality hiPSCs, capable of maintaining their differentiation potential. Pluripotency of the hiPSCs was maintained as demonstrated by subsequent non-directed differentiation (EB formation) and directed differentiation (melanocyte differentiation). Directed differentiation of PBMC418iPS1506 hiPSCs into melanocytic cells. The differentiated derivatives (PBMC418iPS1506_Mel Diff) expressed melanocytic biomarkers MITF and MART-1. For complete article see: Lonza Resource Notes, Fall 2014, page 8-11. 7 BioResearch Stem Cell Products Pluripotent Stem Cells – Proliferation L7™ hPSC Culture System “How can I feed my hPSCs every other day while still maintaining normal karyotype and differentiation potential?” The L7™ hPSC Culture system is a complete system for the maintenance of human ESCs and iPSCs. It is a xeno-free, fully defined, robust system that reduces the PSC workload by supporting every other day feeding of hPSCs. The L7™ hPSC Culture System supports efficient differentiation while maintaining pluripotency marker expression, karyotype and morphology for over 40+ passages with various hiPSC and hESC lines. The L7™ hPSC Culture System also allows for easy transition to clinical applications. This same system is used by Lonza’s Services group to create clinical grade master cell banks for various clinical institutions around the world. SOLUTION: Lonza now offers the L7™ hPSC Culture System - a xeno-free, defined system that allows for every other day feeding of hPSCs. L7™ hPSC Basal Medium and Medium Supplement Lonza took a different approach when developing its hPSC culture system. Instead of using DMEM-F/12, we decided to design a basal medium specifically for culturing PSCs. We then used an experimental approach to assemble the optimal combination and concentrations of growth factors and cytokines that support the long-term growth and maintenance of these undifferentiated PSCs. L7™ hPSC Matrix Lonza’s recombinant hPSC substrate is xeno-free and defined. When used in conjunction with L7™ hPSC Medium, it is proven to support efficient attachment, maintain pluripotency and a stable karyotype for over 40+ passages in multiple hPSC lines. L7™ hPSC Passaging Solution Lonza’s L7™ hPSC Passaging Solution is a chemically-defined, nonenzymatic cell detachment formulation based on a hypertonic sodium citrate solution. It gently dislodges multicellular colony fragments without the need for mechanical manipulation of the cultures. This user friendly method simplifies routine maintenance of your valuable hESC and iPSC lines. L7™ hPSC Cryosolution Lonza’s cryopreservation cocktail supports efficient recovery of cryopreserved hPSCs under xeno-free and defined conditions. 8 BioResearch Stem Cell Products Pluripotent Stem Cells – Proliferation Case Study An hPSC Culture System for Research and Future Clinical Applications By Ashish Mehta1, Chrishan JA Ramachandra1, Glen L Sequiera1, Scott D’Andrea2, Amber Ellis2, Winston Shim1,3 1) National Heart Research Institute Singapore, Singapore; 2) Lonza Walkersville, Inc., Walkersville, MD, USA; 3) DUKE-NUS, Singapore The L7™ hPSC Culture System was evaluated by researchers at the National Heart Research Institute in Singapore by using human embryonic stem cells and lab generated induced pluripotent stem cells. After four passages in culture it was determined L7™ hPSC Culture System is a robust xeno-free and defined system that allows for efficient proliferation of hPSCs. A B l ll lll (A) Schematic representation of dissociation protocol using L7 ™ hPSC Passaging Solution. (B) Phase contrast images showing stages of hPSC after addition of L7 ™ hPSC Passaging Solution for 5 minutes (l) note disruption of hPSC colonies (ll) uniform small cell aggregates after Step 2 (lll) and colonies after 16 hours of seeding on a fresh L7 ™ hPSC Matrix Coated Plate in L7 ™ hPSC Medium. See Lonza’s 2014 Fall Resource Notes article (page 5-7) for additional data and full article. 9 BioResearch Stem Cell Products Pluripotent Stem Cells – Differentiation and Modification Pluripotent Stem Cell-derived Cell Types Lonza and its collaborators utilize proprietary methods for scalable production of physiologically relevant cell types from pluripotent stem cells. These highly pure cell populations specifically address issues of limited tissue availability, donor and preparation variability, and improve the predictive strength of current animal and cell based models. Benefits – Pure human cells; no species extrapolation necessary – Available in large quantities – Avoid difficult isolations, poor yields, low purity and / or lengthy differentiation processes – Minimal lot-to-lot variation – Ready-to-use cryopreserved vials for more flexibility in your experimentation Standard MotorPlate™ Cells stained for Neuronal Class III β-tubulin (green) and Glial Fibrillary Acid Protein (red). Nuclei are counterstained with DAPI (blue). Human ESC-derived Motor Neuron Progenitors Cryopreserved MNPs – MNPs frozen down at approximately day 28 after induction of neural differentiation. Applications – Viability, toxicity, and neuronal outgrowth assays – Maturation studies and functional testing – Motor neuron disease research (ALS, SMA, etc.) Cell Testing and Specifications – 75% Neuronal Class III β-tubulin (TUJ1) – 30% Neurofilament (SMI-32) – mature only – 15% of Glial Fibrillary Acid Protein (GFAP) 10 Pluripotent Stem Cells – Differentiation and Modification Genome Editing using the Nucleofector™ Technology “How can I stably modulate gene expression in pluripotent stem cells?” For genome editing, engineered nucleases are transferred into cells to delete, insert or replace a targeted genomic sequence. Those nucleases are fused or interact with sequence specific DNA binding components directing the nuclease to the target sequence. SOLUTION: Our non-viral Nucleofector™ Technology allows simple and reliable genome editing of ESC's, iPSCs or other cells using ZFN, TALEN or CRISPR/ Cas9 technologies. Lonza’s non-viral Nucleofector™ Technology has been shown to work as a reliable and sufficient method for transferring the required DNA- or RNA-based components into various cell lines and primary or stem cells, like e.g. primary T cells, human ES or iPS cells. Nucleofection Benefits for Genome Editing – High transfection efficiencies for a broad range of cell types, including iPSC – Efficient co-transfection of various substrates – Same conditions for transfecting plasmid DNA, mRNA or PCR cassettes, ssODN – Proven for ZFN, TALEN and CRISPR by more than 30 publications, including high ranking journals www.lonza.com /genome-editing – for further information Transfection Results for Pluripotent Stem Cells Efficiency* Viability* Kit for 4D-Nucleofector™ and 96-well Shuttle™ Systems Kit for Nucleofector™ II/2b Device Embryonic Stem Cells, human (general) 20–78% 50–98% P3 Primary Cell Human Stem Cell Starter Embryonic Stem Cells, human (H1) 22% 96% Primary Cell Optimization Human Stem Cell 1 Embryonic Stem Cells, human (H9) 51–57% 48–83% Primary Cell Optimization Human Stem Cell 2 Embryonic Stem Cells, human (H9.2) 45–78% 57–72% Primary Cell Optimization Human Stem Cell 1 Embryonic Stem Cells, human (Hues-9) 30–55% 50–75% Primary Cell Optimization Human Stem Cell 2 Embryonic Stem Cells, human (BG01V) 20–30% Cell Line Optimization Human Stem Cell 2 Embryonic Stem Cells, mouse (mESC) 50–90% 68–99% P3 Primary Cell Mouse ES Induced Pluripotent Stem Cell, human 50–60% 50–75% P3 or P4 Primary Cell Human Stem Cell Starter Cells marked blue have Lonza-validated optimized protocols. *Approximate ranges extrapolated from larger result collections, including Lonza and customer data. www.lonza.com /celldatabase – for further information and protocol guidance 11 BioResearch Stem Cell Products Adult Stem Cells – Proliferation Choose from Lonza’s extensive range of isolated primary stem cells and culture them in medium optimized for growth or differentiation. Avoid costly and variable isolations. Lonza's adult stem cells are high purity and possess full differentiation capacity. “How can I get the full differentiation potential from my stem cells?” SOLUTION: Lonza stem cells have high purity and homogeneity (confirmed by FACS analysis of positive and negative markers). New and Improved Cell Characterization of MSCs to Support ISCT Standards. Human Mesenchymal Stem Cells – – – – Isolated from bone marrow >90% positive for CD105, CD166, CD44, CD90, CD73 <5% negative for CD34, CD45, CD14, CD19, HLA-DR Tested for the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages Functional assays available to quantify intracellular lipid accumulation (AdipoRed™ Assay Reagent) and lipolysis of differentiated adipocytes (AdipoLyze™ Lipolysis Detection Assay) Functional assay to quantify in vitro mineralization by differentiated osteoblasts (OsteoImage™ Mineralization Assay) Negative for HIV-1, Hepatitis-B, and Hepatitis-C All cells test negative for bacterial, fungal, and mycoplasma contamination – – – – MSC Media Differentiation Kits are exclusively licensed by Lonza from Osiris Therapeutics, Inc. and are subject to the following limited use license: The included biological material, including progeny and derivatives, (collectively referred to as material) is licensed to you under specific terms. You are responsible for ensuring that the terms of the license agreement are met. Grants of license: Lonza Walkersville, Inc. grants you a non-transferable, non-exclusive license to use the material for research. Not for human use: The material may not be used: a) in humans; b) in conjunction with human clinical trials; c) in association with human diagnostics. Material not transferable: You may not transfer the material to any other person or organization. Patent notice: Material under license from Osiris Therapeutics, Inc. Material is covered by US patent 5,486,359 and others. Forward Scatter hMSC differentiated chondrocytes hMSC differentiated osteoblasts 1,000 1,000 800 800 600 600 400 400 200 200 0 200 400 0 Side Scatter 600 800 1,000 0 hMSC differentiated adipocytes 200 400 0 Side Scatter 600 800 1000 Poietics™ hMSC-human Mesenchymal Stem Cells showing a uniform profile analyzed by flow cytometry, indicating a pure population. 12 Adult Stem Cells – Proliferation Rat Mesenchymal Stem Cells Human CD34+ Cells – Positive for CD29, CD90 – Negative for CD11b, CD34, CD45 – Tested for the ability to differentiate into osteogenic and adipogenic lineages – All cells test negative for bacterial, fungal, and mycoplasma contamination – Isolated from bone marrow and cord blood – Isolated from mononuclear cells using positive immunomagnetic selection – Cell purity is > 90% via FACS – Negative for HIV-1, Hepatitis-B, and Hepatitis-C – All cells test negative for bacterial, fungal, and mycoplasma contamination HPGM™ Hematopoietic Growth Medium Rat MSCs stained with OsteoImage™ Assay for bone mineralization (osteogenesis) A chemically defined, serum-free, xeno-free media developed to support (with the addition of appropriate cytokines) the proliferation and differentiation of CD34+ hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC). HPGM™ Hematopoietic Growth Medium does not contain any exogenous growth factors, artificial stimulators of cellular proliferation, or undefined supplements. Rat MSCs differentiated into adipocytes stained with Oil Red O Human Adipose-derived Stem Cells – – – – Isolated from lipoaspirate Positive for CD13, CD29, CD44, CD73, CD90, CD105, CD166 Negative for CD14, CD31, CD45, HIV-1, Hepatitis-B, and Hepatitis-C Functional assays available to quantify intracellular lipid accumulation (AdipoRed™ Assay Reagent) and lipolysis of differentiated adipocytes (AdipoLyze™ Lipolysis Detection Assay) – All cells test negative for bacterial, fungal, and mycoplasma contamination Dental pulp stem cells stained for dentin sialophosphoprotein (DSPP) (red) and DAPI (blue) Human Dental Pulp Stem Cells ADSC – derived adipocytes stained with Oil Red O ADSC – chondrocytes stained with Alcian Blue – Poietics™ Human Dental Pulp Stem Cells are mesenchymal like stem cells harvested from human teeth – Isolated adult third molars – Positive for CD105, CD166, CD29, CD90, CD73 – Negative for CD133, CD34, CD45 – Cell purity is > 90% via flow cytometry – Negative for HIV-1, Hepatitis-B, and Hepatitis-C – All cells test negative for bacterial, fungal, and mycoplasma contamination ADSC – derived osteoblasts stained with Alizarin Red 13 BioResearch Stem Cell Products Adult Stem Cells – Proliferation Preadipocytes Osteoclast Precursors – Poietics™ Subcutaneous Preadipocytes are precursor cells, isolated from subcutaneous fat, that develop into adipocytes when fully differentiated and are characterized by the accumulation of intracellular triglycerides – Poietics™ Human Visceral Preadipocytes are precursor cells, isolated from visceral, abdominal tissue (kidney and bladder), that develop into adipocytes when fully differentiated and are characterized by the accumulation of intracellular triglycerides – Characterized morphologically at day 10, using AdipoRed™ Assay – Negative for HIV-1, Hepatitis-B, and Hepatitis-C – All cells test negative for bacterial, fungal, and mycoplasma contamination – Characterized morphologically at day 7 as large, multi-nucleated cells – Differentiated cells stain positive for tartrate-resistant acid phosphatase (TRAP) – Functional assay to quantify in vitro bone resorption by differentiated osteoclasts (OsteoLyse™ Assay Kit, CalciFluor™ Bone Resorption Assay, OsteoAssay™ Human Bone Plate) – Negative for HIV-1, Hepatitis-B, and Hepatitis-C – All cells test negative for bacterial, fungal, and mycoplasma contamination Differentiated subcutaneous preadipocyte cells Differentiated human osteoclasts resorbing bone fragment Differentiated visceral preadipocyte cells stained with oil red O Neural Progenitors – Neurospheres differentiate into a mixed population of neurons and astrocytes – Following adhesion to laminin in the presence of brain-derived neurotrophic factor (BDNF, 25 ng/mL), differentiated NHNP stain positive for beta tubulin III, specific for neuronal lineage, and Glial Fibrillary Acidic Protein (GFAP), denoting astrocyte lineage. – Negative for HIV-1, Hepatitis-B, and Hepatitis-C – All cells test negative for bacterial, fungal, and mycoplasma contamination NHNP stained for ß-tubulin III and GFAP 14 Pits formed from bone resorption activity of differentiated osteoclasts Adult Stem Cells – Differentiation and Modification Nucleofector™ Technology for Transfection of Adult Stem Cells % Transfection efficiency/viability Comparison of Nucleofection with Lipofection for Transfection of Human MSC Nucleofector™ Technology is the efficient non-viral transfection method for pluripotent and adult stem cells, as well as their differentiated progenitors. Suited for almost any substrate, Nucleofection enables efficient transfection without altering the biochemical functionality of the transfected cell. – Optimized transfection protocols for a variety of stem and progenitor cells – Preserved cellular functionality and reduced toxicity, maintaining differentiation potential – Different device platforms supporting broad spectrum of cell numbers and throughput 110 100 90 80 70 60 50 40 30 20 10 0 Nucleofection Transfection efficiency Reagent F Reagent D Viability MSC were transfected by Nucleofection with pcDNA3/NT-GFP using either Nucleofection or the lipid-based. MSC transfected by Nucleofection were analyzed for transfection efficiency roughly 60 hours post Nucleofection, cells transfected with lipid reagents were analyzed after 72 hours. Transfection efficiency was scored by flow cytometric analysis and reported as percentage of GFP+ cells. The percentage of viable cells was estimated by trypan blue exclusion. (Data courtesy of Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, Chiodoni C, Colombo MP, Versura P, D’Errico-Grigioni A, Ferri E, Baccarani M and Lemoli RM, Institute of Hematology and Medical Oncology, Bologne, Italy). Transfection Results for Pluripotent Stem Cells Efficiency* Viability* Kit for 4D-Nucleofector™ and 96-well Shuttle™ Kit for Nucleofector™ II/2b Device Systems Adipose-derived stem cells, human (ADSC) 73–94% 58–85% P1 Primary Cell Human MSC CD34+ cells, human 83% 62–70% P3 Primary Cell Human CD34 Mesenchymal stem cells, human (hMSC) 40–88% 50–86% P3 Primary Cell Human MSC Preadipocytes, subcutaneous, human 31–84% 33–95% P1 Primary Cell Basic Fibroblast Preadipocytes, visceral, human 28–94% 35–90% P1 Primary Cell Basic Fibroblast Neural precursor, cow 62–65% 73–75% Primary Cell Optimization Basic Neuron Neural stem cell, human (hNSC) 90% Primary Cell Optimization Mouse NSC Neural stem cell, mouse (mNSC) 60–82% 80% P4 Primary Cell Mouse NSC Primary Cell Optimization Rat NSC 90% P3 Primary Cell Basic Glial Cell Primary Cells Neural stem cell, rat (rNSC) 42–46% Oligodendrocyte precursors, rat 17% Cells marked blue have Lonza-validated optimized protocols. *Approximate ranges extrapolated from larger result collections, including Lonza and customer data. www.lonza.com /celldatabase – for further information and protocol guidance 15 BioResearch Stem Cell Products Cell Analysis Tools – Live Cell Imaging CytoSMART™ System Your Stem Cell Culture is Just One Click Away Monitor your stem cell culture anytime using our new CytoSMART™ System – your personal live cell imaging device. The System consists of a mini-microscope, the CytoSMART™ Device, that is linked to an accompanying tablet outside the incubator. Via this tablet you start your CytoSMART™ Projects and capture the images of your cell culture in defined intervals. All images are transmitted to the CytoSMART™ Connect Cloud via wireless transmission (Wifi), meaning, you can monitor your cell culture remotely and in real-time with any browsercapable device, be it your smartphone, tablet or PC. The Smarter Way for Stem Cell Cultures With the CytoSMART™ System your cells can remain in their defined conditions in the incubator while you monitor the stem cell culture online. – Easy documentation and video-taping of your stem cell culture, e.g. for generation of iPSC – Remote monitoring of stem cell cultures grown under hypoxic conditions or under GMP condition – Cell culture standardization through operator independent determination of confluency with automatic alerts for cell culture functionality – Easy and immediate download of cell culture images or videos from CytoSMART™ Connect Cloud Image of iPSC culture maintained in L7™ System using CytoSMART™ Lux 10X System. Please refer to our website to view a video of an iPSC culture taken with the CytoSMART™ System. www.lonza.com /cytosmart 16 Cell Analysis Tools – Assays OsteoAssay™ Human Bone Plate Utilize Lonza’s research tools for every step of your workflow. Lonza provides everything you need for biologically relevant results – from culture media and sample preparation, all the way through protein confirmation. The OsteoAssay™ Plate provides a thin layer of adherent real human bone for the culture of primary human or non-human osteoclasts, osteoclast precursors, or immortalized cell lines. Cells can be stained with standard cytochemical or immunofluorescent techniques. Assays of bone resorption and osteoclast precursor differentiation can be performed easily by sampling the cell culture supernatant. AdipoLyze™ Lipolysis Detection Assay The AdipoLyze™ Lipolysis Detection Assay is a fast, sensitive, fluorescent in vitro assay used for detecting small quantities of glycerol in cells undergoing lipolysis. OsteoImage™ Mineralization Assay The OsteoImage™ Mineralization Assay is a rapid, fluorescent in vitro assay for assessing bone cell mineralization. The OsteoImage™ Assay can quantitate in vitro mineralization by osteogenic stem cells, primary osteoblasts, and osteoblast-like cell lines. It is based on specific binding of the fluorescent OsteoImage™ Staining Reagent to the hydroxyapatite portion of bone-like nodules deposited by cells. Unlike typical histochemical methods such as von Kossa and Alizarin red, neither of which is hydroxyapatite specific, the OsteoImage™ Assay eliminates multiple steps or tedious extraction steps. AdipoRed™ Assay Reagent The AdipoRed™ Assay Reagent is designed for assessing the effect of compounds on the differentiation of preadipocytes or on lipid utilization in mature adipocytes. This objective, high-throughput, homogeneous fluorescence-based assay quantifies the accumulation of intracellular triglycerides and provides significant advantages to drug discovery efforts. ToxiLight™ Kit OsteoLyse™ Assay Kit Check a compound’s cytotoxic effects non-destructively by measuring adenylate kinase (AK) leakage from damaged cells. The OsteoLyse™ Assay Kit provides an easy to use protocol for quantitatively measuring in vitro osteoclast-mediated bone resorption in a highthroughput format. Osteoclasts can be seeded onto the OsteoLyse™ Plate using traditional cell culture protocols. The assay directly measures the release of Europium-labeled collagen fragments into the osteoclast cell culture supernatant via time resolved fluorescence, indicating their resorption activity levels. ViaLight™ Plus Kit For a fast, sensitive and safe measurement of cytotoxicity and cell proliferation. MycoAlert™ Mycoplasma Detection Assay The MycoAlert™ Assay Kit is a selective biochemical test that exploits the activity of specific mycoplasmal enzymes which are found in all 6 of the main mycoplasma cell culture contaminants and the vast majority of 180 species of mycoplasma. 17 BioResearch Stem Cell Products Cell Analysis Tools – Electrophoresis Custom Research Solutions Molecular Biology Products If you’re looking for customized research solutions or expert support services, just ask your local Lonza representative. They can put you in touch with experts who have decades of experience in cell isolation, transfection, and testing services. Nucleic Acid Electrophoresis – FlashGel™ System Protein Electrophoresis – PAGEr™ EX Gels – ProSieve™ EX Reagents Custom Cell Services Lonza’s molecular biology products offer basic aspects of cancer research. Speed up your results from PCR analysis, RNA integrity analysis, to fragment isolation for cloning. Lonza’s FlashGel™ System can get you there in minutes. For protein analysis and western blotting Lonza’s PAGEr™ EX Gels and ProSieve™ EX Reagents help speed your research from hours to minutes. You can order virtually any human or animal cell through our vast tissue network. Lonza’s isolation experts can provide you with: – A broad range of cells, packaged to your specifications – Customized treatments, such as cell pelleting, personalized media, and specific treatment prior to cryopreservation Scientific Support For advice or information on any Lonza product, just contact our scientific support team. They offer: – More than 50 years of combined troubleshooting experience – Educational webinars – Real disease research application data You can find contact details for your regional support team on the back of this brochure. 18 Ordering Information: Primary Cells and Media Cat. No. Product Name Size L7™ hiPSC Reprogramming and hPSC Culture System L7™ hPSC Culture System FP-5013 L7™ hPSC Passaging Solution 100 mL FP-5002 L7™ hPSC Cryosolution 50 mL FP-5020 L7™ hPSC Matrix 1 mg FP-5007 L7™ hPSC Medium 500 mL basal medium + 5 mL supplement L7™ PBMC Reprogramming Bundle FP-5124 L7™ PBMC Priming-Recovery Kit, contains: L7™ PBMC Priming-Recovery Basal Medium and L7™ PBMC Reprogramming Enhancers A and B 24 rxn CC-2702 hPBMCs – Human Peripheral Blood Mononuclear Cells ≥ 50 million cells V4XP-3024 P3 Primary Cell 4D-Nucleofector™ Kit 24 rxn (100 µl Nucleocuvette™) Adult Stem Cells and Media Human Mesenchymal Stem Cells PT-2501 hMSC – Human Mesenchymal Stem Cells, cryopreserved ≥750,000 cells PT-3001 MSCGM™ Mesenchymal Stem Cell Growth Medium BulletKit™, contains 1 ea. PT-3238 and PT-4105 Kit PT-3002 hMSC – Human Mesenchymal Stem Cell Osteogenic Differentiation Medium BulletKit™, contains 1 ea. PT-3924 and PT-4120 Kit PT-3003 hMSC – Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium BulletKit™, contains 1 ea. PT-3925 and PT-4121 Kit PT-3004 hMSC – Human Mesenchymal Stem Cell Adipogenic Differentiation Medium BulletKit™, contains 1 ea. PT3102B, 3102A, 4122 and 4135 Kit PT-3238 MSCBM™ – Mesenchymal Stem Cell Basal Medium 440 mL PT-4105 MSCGM™ Mesenchymal Stem Cell Growth Medium SingleQuots™ Supplements and Growth Factors Kit PT-4124 rhTGF-β3 for hMSC Chondrogenic Differentiation Medium Supplement 2 μg 190620 MSCGM™ CD Mesenchymal Stem Cell Basal Medium 500 mL 190632 MSCGM™ CD Mesenchymal Stem Cell Growth Medium BulletKit™, chemically defined, with L-glutamine, without phenol red and antibiotics Kit 192125 MSCGM™ CD Mesenchymal Stem Cell Growth Medium SingleQuots™, supplements and growth factors Kit Human Adipose-Derived Stem Cells PT-3273 ADSC – Apidose-Derived Stem Cells Basal Medium 500 mL PT-4503 ADSC – Apidose-Derived Stem Cells Growth Medium SingleQuots™ Supplements and Growth Factors Kit PT-4505 ADSC – Apidose-Derived Stem Cells Growth Medium BulletKit™, includes PT-4503 and PT-3273 Kit PT-5006 Human Adipose-Derived Stem Cells ≥1 million cells PT-5007 D-HADSC – Diseased Human Adipose-derived Stem Cells – Diabetes Type I ≥1 million cells PT-5008 D-HADSC – Diseased Human Adipose-derived Stem Cells – Diabetes Type II ≥1 million cells Human Dental Pulp Stem Cells PT-5025 hDPSC – Human Dental Pulp Stem Cells ≥1 million cells PT-3927 DPSC – Dental Pulp Stem Cell Basal Medium 500 mL PT-4516 DPSC – Dental Pulp Stem Cell SingleQuots™ Supplements and Growth Factors Kit PT-3005 DPSC – Dental Pulp Stem Cell BulletKit™ Medium Kit Rat Mesenchymal Stem Cells PT-2505 R-MSC – Rat Mesenchymal Stem Cells ≥750,000 cells 192853 Rat MSCGM™ Mesenchymal Stem Cell Growth Medium BulletKit™ Kit 192855 rMSC – Rat Mesenchymal Stem Cell Adipogenic Differentiation BulletKit™ Kit 19 Ordering Information: Primary Cells and Media Cat. No. Product Name Size Progenitor Precursor Cells and Media Human CD34+ Cells 1M-101 Human Bone Marrow CD34+ Progenitor Cells, fresh ≥100,000 cells 1M-101A Human Bone Marrow CD34+ Progenitor Cells, fresh ≥300,000 cells 1M-101B Human Bone Marrow CD34+ Progenitor Cells, fresh ≥500,000 cells 1M-101C Human Bone Marrow CD34+ Progenitor Cells, fresh ≥1 million cells 2M-101 Human Bone Marrow CD34+ Progenitor Cells, cryopreserved ≥100,000 cells 2M-101A Human Bone Marrow CD34 Progenitor Cells, cryopreserved ≥300,000 cells 2M-101B Human Bone Marrow CD34+ Progenitor Cells, cryopreserved ≥500,000 cells 2M-101C Human Bone Marrow CD34+ Progenitor Cells, volume discount available ≥1 million cells 2M-101D Human Bone Marrow CD34+ Progenitor Cells, cryopreserved ≥2 million cells 2M-101F Human Bone Marrow CD34+ Progenitor Cells, cryopreserved ≥10 million cells 2C-101 Human Cord Blood CD34+ Progenitor Cells, cryopreserved ≥1 million cells 2C-101A Human Cord Blood CD34 Progenitor Cells, cryopreserved ≥500,000 cells 2C-101B Human Cord Blood CD34+ Progenitor Cells ≥100,000 cells PT-3926 HPGM™ Hematopoietic Progenitor Growth Medium 500 mL + + Human Preadipocyte Cells PT-5001 Human Subcutaneous Preadipocyte Cells, cryopreserved ≥4 million cells PT-5005 Human Visceral Preadipocyte Cells, cryopreserved ≥1 million cells PT-5020 Human Subcutaneous Preadipocyte Cells, cryopreserved ≥1 million cells PT-5021 Diseased Human Subcutaneous Preadipocyte Cells, Diabetes Type I, cryopreserved ≥1 million cells PT-5022 Diseased Human Subcutaneous Preadipocyte Cells, Diabetes Type II, cryopreserved ≥1 million cells PT-5023 Diseased Human Visceral Preadipocyte Cells, Diabetes Type I, cryopreserved ≥1 million cells PT-5024 Diseased Human Visceral Preadipocyte Cells, Diabetes Type II, cryopreserved ≥1 million cells PT-8002 PGM™ 2 Preadipocyte Growth Medium-2 BulletKit™, contains 1 ea. PT-8202, PT-9502 Kit PT-8202 PBM™ 2 Preadipocyte Basal Medium-2 500 mL PT-9502 PGM™ 2 Preadipocyte Growth Medium-2 SingleQuots™ Supplements and Growth Factors Kit Human Osteoclast Precursor Cells 2T-110 hOPC – Human Osteoclast Precursor Cells, cryopreserved ≥1 million cells PT-8001 OCP – Osteoclast Precursor Medium BulletKit™, contains 1 ea. PT-8201 and PT-9501 Kit PT-8201 OCP – Osteoclast Precursor Basal Medium 100 mL PT-9501 OCP – Osteoclast Precursor Medium SingleQuots™ Supplements and Growth Factors Kit Human Neural Progenitor Cells PT-2599 NHNP – Human Neural Progenitor Cells ≥1.2 million cells CC-3209 NPMM™ Neural Progenitor Maintenance Medium BulletKit™, contains 1 ea. CC-3210, CC-4241 and CC-4242 Kit CC-3210 NPBM™ Neural Progenitor Basal Medium 200 mL CC-3229 NPDM™ Neural Progenitor Differentiation Medium BulletKit™, contains 1 ea. CC-3210 and CC-4242 Kit CC-4241 NPMM™ Neural Progenitor Maintenance Medium SingleQuots™ Supplements and Growth Factors Kit CC-4242 Neural Progenitor Supplement SingleQuots™ Supplements and Growth Factors Kit 20 Continued on next page Ordering Information: Transfection Choose the Nucleofection Platform that Suits Your Research Needs Advanced Platform 96-well Add-on High-throughput Platform 4D-Nucleofector™ System 96-Well Shuttle™ System 384-Well Nucleofector™ System Nucleofector™ 2b Device Throughput (samples per run) Low to medium (1–16) Low to high (1–96) High (384) Low (1) Reaction volume 20 µL + 100 µL 20 µL 20 µL 100 µL Electrode material Conductive polymer Conductive polymer Conductive polymer Aluminum Low cell numbers (20 µL) 5 × 104 to 5 × 105 5 × 104 to 5 × 105 5 × 104 to 5 × 105 – High cell numbers (100 µL) 4–5 × 106 DNA vector amount/sample 0.2–1 μg (20 µL) 1–5 μg (100 µL) 0.2–1 μg siRNA amount/sample (concentration 2 nM – 2 µM) 0.04–40 pmol (20 µL) 0.2–200 pmol (100 µL) 0.04–40 pmol Device Basic Device Unit 4–5 × 106 0.2–1 μg 1–5 μg 0.04–40 pmol 0.2–200 pmol Adherent Nucleofection – – – Compatibility with 96-well Shuttle™ System – – – Cat. No. Product Name Size Nucleofector™ Devices AAB-1001 Nucleofector™ 2b Device Device AAF-1001B 4D-Nucleofector™ Core Unit Device AAF-1001X 4D-Nucleofector™ X Unit Device AAM-1001S 96-well Shuttle™ Device Device Kits for 4D-Nucleofector™ Device V4XP-1012 P1 Primary Cell 4D-Nucleofector™ X Kit L 12 rxn (100 µL Nucleocuvette™) V4XP-1024 24 rxn (100 µL Nucleocuvette™) V4XP-1032 P1 Primary Cell 4D-Nucleofector™ X Kit S 32 rxn (20 µL Nucleocuvette™; 16-well) V4XP-3012 P3 Primary Cell 4D-Nucleofector™ X Kit L 12 rxn (100 µL Nucleocuvette™) V4XP-3024 24 rxn (100 µL Nucleocuvette™) V4XP-3032 P3 Primary Cell 4D-Nucleofector™ X Kit S 32 rxn (20 µL Nucleocuvette™; 16-well) V4XP-4012 P4 Primary Cell 4D-Nucleofector™ X Kit L 12 rxn (100 µL Nucleocuvette™) V4XP-4024 P4 Primary Cell 4D-Nucleofector™ X Kit L 24 rxn (100 µL Nucleocuvette™) V4XP-4032 P4 Primary Cell 4D-Nucleofector™ X Kit S 32 rxn (20 µL Nucleocuvette™; 16-well) V4XP-9096 Primary Cell Optimization 4D-Nucleofector™ X Kit 96 rxn (20 µL Nucleocuvette™; 16-well) Kits for 96-well Nucleofector™ Device V4SP-1096 P1 Primary Cell 96-well Nucleofector™ Kit 96 rxn (20 µL Nucleocuvette™) V4SP-1960 V4SP-3096 960 rxn (20 µL Nucleocuvette™) P3 Primary Cell 96-well Nucleofector™ Kit 96 rxn (20 µL Nucleocuvette™) V4SP-3960 V4SP-4096 960 rxn (20 µL Nucleocuvette™) P4 Primary Cell 96-well Nucleofector™ Kit 96 rxn (20 µL Nucleocuvette™) V4SP-4960 V4SP-9096 960 rxn (20 µL Nucleocuvette™) Primary Cell Optimization 96-well Nucleofector™ Kit 160 rxn (20 µL Nucleocuvette™) 21 BioResearch Stem Cell Products Ordering Information: Transfection Cat. No. Product Name Size Kits for Nucleofector™ II/2b Device VACA-1003 Cell Line Nucleofector™ Kit V 10 rxn (100 µL Aluminium cuvette) VCA-1003 25 rxn (100 µL Aluminium cuvette) VVCA-1003 VAPA-1003 100 rxn (100 µL Aluminium cuvette) Human CD34+ Cell Nucleofector™ Kit 10 rxn (100 µL Aluminium cuvette) VPA-1003 25 rxn (100 µL Aluminium cuvette) VVPA-1003 VAPG-1004 100 rxn (100 µL Aluminium cuvette) Mouse Neural Stem Cell Nucleofector™ Kit 10 rxn (100 µL Aluminium cuvette) VPG-1004 25 rxn (100 µL Aluminium cuvette) VVPG-1004 VAPG-1005 100 rxn (100 µL Aluminium cuvette) Rat Neural Stem Cell Nucleofector™ Kit 10 rxn (100 µL Aluminium cuvette) VPG-1005 25 rxn (100 µL Aluminium cuvette) VVPG-1005 VAPE-1001 100 rxn (100 µL Aluminium cuvette) Human Mesenchymal Stem Cell Nucleofector™ Kit 10 rxn (100 µL Aluminium cuvette) VPE-1001 25 rxn (100 µL Aluminium cuvette) VVPE-1001 VAPH-1001 100 rxn (100 µL Aluminium cuvette) Mouse Embryonic Stem Cell Nucleofector™ Kit 10 rxn (100 µL Aluminium cuvette) VPH-1001 25 rxn (100 µL Aluminium cuvette) VVPH-1001 100 rxn (100 µL Aluminium cuvette) VPH-5002 Human Stem Cell Nucleofector™ Starter Kit 18 rxn (100 µL Aluminium cuvette) VAPH-5012 Human Stem Cell Nucleofector™ Kit 1 10 rxn (100 µL Aluminium cuvette) VPH-5012 25 rxn (100 µL Aluminium cuvette) VVPH-5012 VAPH-5022 100 rxn (100 µL Aluminium cuvette) Human Stem Cell Nucleofector™ Kit 2 10 rxn (100 µL Aluminium cuvette) VPH-5022 25 rxn (100 µL Aluminium cuvette) VVPH-5022 VAPI-1002 100 rxn (100 µL Aluminium cuvette) Basic Nucleofector™ Kit for Primary Mammalian Neurons (for cow neural progenitors) VPI-1002 25 rxn (100 µL Aluminium cuvette) VVPI-1002 VAPI-1003 10 rxn (100 µL Aluminium cuvette) 100 rxn (100 µL Aluminium cuvette) Basic Nucleofector™ Kit for Primary Mammalian Neurons (for cow neural progenitors) 10 rxn (100 µL Aluminium cuvette) VPI-1003 25 rxn (100 µL Aluminium cuvette) VVPI-1003 100 rxn (100 µL Aluminium cuvette) 22 Ordering Information: Cell Analysis Cat. No. Product Name Size Cell Analysis BioAssays for Cell Proliferation and Cytotoxicity LT07-117 ToxiLight™ Non-Destructive Cytotoxicity BioAssay Kit 1,000 tests LT07-121 ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay Kit 1,000 tests LT07-122 ViaLight™ MDA Plus Microbial Proliferation and Cytotoxicity BioAssay Kit 1,000 tests LT07-217 ToxiLight™ Non-Destructive Cytotoxicity BioAssay Kit 500 tests LT07-221 ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay Kit 500 tests LT07-321 ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay Kit 10,000 tests LT07-517 ToxiLight™ 100% Lysis Control Set 10 mL, 200 tests (sold separately) LT17-217 ToxiLight™ Non-Destructive Cytotoxicity BioAssay Kit, with plates 500 tests LT17-221 ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay Kit, with plates 500 tests BioAssays for Cell Function 193339 AdipoLyze™ Lipolysis Detection Assay 96 tests PA-1000 OsteoAssay™ Human Bone Plate 96-well plate PA-1500 OsteoLyse™ Assay Kit, human collagen 96 tests PA-1503 OsteoImage™ Mineralization Assay 480 tests PT-7009 AdipoRed™ Assay Reagent 5 × 4.0 ml CytoSMART™ System AACS-1002 CytoSmart™ Lux 10X System Device 23 www.lonza.com/research www.lonza.com /stemcells Contact Information North America Customer Service: +1 800 638 8174 (toll free) order.us@lonza.com Scientific Support: +1 800 521 0390 (toll free) scientific.support@lonza.com Europe Customer Service: +32 87 321 611 order.europe@lonza.com Scientific Support: +32 87 321 611 scientific.support.eu@lonza.com International Contact your local Lonza distributor Customer Service: +1 301 898 7025 Fax: +1 301 845 8291 scientific.support@lonza.com International Offices Australia Belgium Brazil France Germany India Japan Luxemburg Singapore The Netherlands United Kingdom + 61 3 9550 0883 + 32 87 321 611 + 55 11 2069 8800 0800 91 19 81 (toll free) 0800 182 52 87 (toll free) +91 22 4342 4000 + 81 3 6264 0660 +32 87 321 611 + 65 6521 4379 0800 022 4525 (toll free) 0808 234 97 88 (toll free) Lonza Walkersville, Inc. – Walkersville, MD 21793 For research use only. Not for use in diagnostic procedures. The Nucleofector™ Technology is covered by patent and/ or patent pending rights owned by the Lonza or its affiliates. All trademarks belong to Lonza or its affiliates or to their respective third party owners. The information contained herein is believed to be correct and corresponds to the latest state of scientific and technical knowledge. However, no warranty is made, either expressed or implied, regarding its accuracy or the results to be obtained from the use of such information and no warranty is expressed or implied concerning the use of these products. The buyer assumes all risks of use and/or handling. Any user must make his own determination and satisfy himself that the products supplied by Lonza Group Ltd or its affiliates and the information and recommendations given by Lonza Group Ltd or its affiliates are (i) suitable for intended process or purpose, (ii) in compliance with environmental, health and safety regulations, and (iii) will not infringe any third party’s intellectual property rights. ©2015 Lonza. All rights reserved. CD-BR009-1 05/15
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